几十本Human英文生物技术图书all chapter 16.docVIP

Chapter 16 Introducing Cloned Genes into Cultured Mammalian Cells Protocol 1: DNA Transfection Mediated by Lipofection Because a large number of factors affect the efficiency of lipofection, this protocol should be viewed as a starting point for systematic optimization of transfection mediated by lipofecting agents. Once a positive signal has been obtained from a transfected plasmid carrying a standard reporter gene, optimal conditions for transfection can be established by systematic variation of parameters such as the initial cell density, the amount and purity of DNA, the media and serum, and the time of exposure of the cells to the cationic-lipid-DNA complex. Protocol 2: Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs Calcium phosphate forms an insoluble precipitate with DNA, which attaches to the cell surface and is taken into the cells by endocytosis. This protocol is a modified version of a method published by Jordan et al. (1996) who rigorously optimized calcium-phosphate-based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells. The protocol is easily adapted for use with other types of cells, both adherent and nonadherent. Protocol 3: Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA This protocol, as all others involving calcium phosphate transfection, is based on the venerable method of Graham and van der Eb (1973). The procedure is used chiefly to generate stable lines of cells carrying chromosomally integrated copies of the transfected DNA. Protocol 4: Transfection Mediated by DEAE-Dextran: High-efficiency Method DEAE-dextran is generally used to obtain a burst of transient expression of cloned genes after transfection of mammalian cells. Many variants of the technique have been described, all of which seek to maximize the uptake of DNA and to minimize the cytotoxic effects of DEAE-dextran. In the following protocol, cells are